Mechanism of Phosphatidylinositol-Specific Phospholipase C:  Origin of Unusually High Nonbridging Thio Effects

Abstract

Phosphatidylinositol-specific phospholipase C (PI-PLC) has been proposed previously to employ a catalytic mechanism highly reminiscent of that of ribonuclease A (RNase A). Both catalytic sites are comprised of two histidine side chains acting as a general base-general acid pair and a phosphate-activating residue: an arginine in the case of PI-PLC and a lysine in RNase A. Despite these structural similarities, the PI-PLC reaction is slowed 10(5)-fold upon substitution of one of the phosphate nonbridging oxygen atoms with sulfur, whereas a much smaller effect is observed in the analogous RNase A reaction. Here, we report a systematic study of this property in PI-PLC, conducted by means of site-directed chemical modification of a cysteine residue replacing the arginine at position 69. The results show that mutant enzymes featuring bidentate side chains at this position display significantly higher activity, higher thio effects, and greater stereoselectivity than do those with monodentate side chains. The results suggest that the bidentate nature of Arg69 is the origin of the large thio effects and stereoselectivity in PI-PLC. We propose that in addition to binding the phosphate, the function of arginine 69 is to bring the phosphate group and the 2-OH group of inositol into proximity and to induce proper alignment for nucleophilic attack, and possibly to lower the pK(a) of the 2-OH. The results presented here could be important to mechanisms of phosphoryl transfer enzymes in general, suggesting that a major part of thio effects observed in enzymatic phosphoryl transfer reactions can originate from factors other than direct interaction between a side chain and a phosphate group, and caution the use of the absolute magnitude of the thio effect as an indicator of the strength of such interactions.

The Catalytic Role of Aspartate in a Short Strong Hydrogen Bond of the Asp274–His32 Catalytic Dyad in Phosphatidylinositol-specific Phospholipase C Can Be Substituted by a Chloride Ion

“…Previously, WT and a series of mutant PI-PLC enzymes were shown to display the same R p thio effect (k O /k R p ) using 1,2-dipalmitoyl-sn-glycero-3-thiophospho-1-myoinositol as the substrate and DHPC as the detergent, suggest- ing that the chemistry step is rate-limiting under the assay conditions (9,18). If the rate is limited by micelle exchanges, the thio effect should increase as the chemical step slows in the mutant.…”

Engineering a Catalytic Metal Binding Site into a Calcium-Independent Phosphatidylinositol-Specific Phospholipase C Leads to Enhanced Stereoselectivity

“…If Ca 2+ indeed substitutes functionally for the side chain of Arg69, as has been demonstrated in the conservative R69 mutants (R69C-AA and R69C-TAA) (28), the presence of Ca 2+ should restore some of the stereoselectivity lost in R69D. To test this hypothesis, we determined the stereoselectivity of R69D in the presence and absence of Ca 2+ by 31 P NMR.…”

“…The activities of all three mutants involving this residue, D33N/R69D, D33E/ R69D, and D33N/D67E/R69D, are all lower than that of the (28). AA, TAA, PA, and EA stand for the covalently linked functional groups acetamidine, thioacetamidine, propylamine, and ethylamine, respectively.…”

“…All mutations were verified by sequencing. Mutant R69C was constructed previously (28). All mutant proteins were purified as described previously (22,24), except that all but the final dialysis buffer included 1 mM EDTA to prevent metal contamination.…”

“…Both structural information 1 Abbreviations: DAG, sn-1,2-diacylglycerol; DHPC, diaheptanoylsn-glycero-3-phosphocholine; DPPsI, 1,2-dipalmitoyl-sn-glycero-3-(1-thiophospho-1D-myo-inositol); EDTA, ethylenediaminetetraacetic acid; HEPES, N-(2-hydroxyethyl) piperazine-N′-(2-ethanesulfonic acid); HSQC, heteronuclear single quantum coherence; [ 3 H]-PI, L-R-[myoinositol-2-3 H(N)]-phosphatidylinositol; IP, inositol 1-phosphate; IcP, inositol 1,2-cyclic phosphate; NMR, nuclear magnetic resonance; NOESY, nuclear Overhauser enhancement spectroscopy; PI, phosphatidylinositol; PI-PLC, phosphatidylinositol-specific phospholipase C; Tris, tris (hydroxyethyl) aminomethane; WT, wild type. and the results from stereochemical and mutagenesis studies have indicated that these two similarly positioned positively charged moieties play similar catalytic roles: both stabilize the transition state of the phosphoryl transfer reaction via interaction with pro-S oxygen of the phosphodiester moiety and facilitate deprotonation of the 2-OH of the inositol ring (16,18,(27)(28)(29). However, it is not clear whether, and how, the use of Ca 2+ versus Arg could be responsible for the differences in the substrate specificity and the product profile (ratio of the cyclic to acyclic product) between mammalian and bacterial enzymes.…”